Cell regulation is mediated by a wide variety of polypeptides. Historically, few of these polypeptides are produced in sufficient amounts to be isolated and characterized. Even in those situations where particular polypeptides are capable of isolation and characterization, the number of amino acids constituting the polypeptides normally preclude synthesis by conventional polypeptide bond formation in commercially useful amounts.
In the last few years, however, a number of discoveries relating to biotechnology have occurred which at the present time promise opportunities for the detection, isolation and production in commercially useful amounts of naturally occurring proteins, which fulfill a wide variety of cell regulatory functions.
The ability to isolate, characterize and insert a gene into a replicating vector, such as a plasmid or phage, and transform a microorganism with the resulting hybrid has introduced new techniques, i.e., genetic engineering techniques, for the production of macromolecular polypeptides. These techniques not only afford the opportunity to obtain polypeptides in abundance, but allow for study of the polypeptides and use of the polypeptides in regulating cell functions in vitro and in vivo.
Because of the cumbersome nature and difficulties associated with synthesis and existence of introns present in chromosomal DNA, the messenger RNA is frequently the desired route where genetic engineering is involved. In each cell, there is continuously produced a large number of different messenger RNA molecules. Therefore, means must be provided for isolating the messenger RNA of interest from other messenger RNA molecules. Where a messenger RNA of interest is normally produced in only small amounts as compared to the total amount of messenger RNAs, it is frequently desirable, if not necessary, to obtain cells which enhance the amount of messenger RNA of interest present in the cell.
As an alternative to genetic engineering, the ability to culture cells offers an opportunity for the production of a wide variety of polypeptides. By isolating specific cells and establishing a culture, which can be expanded and maintained for extensive periods of time, one can directly produce the polypeptides of interest from the cultured cells. In this manner, one avoids the need to isolate the gene or messenger RNA of interest and perform the numerous complicated steps involved with successful genetic engineering.
The regulation of cell growth is a poorly understood topic. A large number of growth regulatory factors have been described heretofore which can either stimulate or inhibit cell growth. A total understanding of the integration of all the signals a cell receives from these factors has not yet been achieved. While such factors have been isolated from many sources, platelets are known to contain large quantities of a variety of potent growth factors. Platelet-derived growth factor (PDGF) and the transforming growth factors alpha and beta (TGF-alpha, TGF-beta, respectively,) fall into this category. Not much is known concerning the physiological function of these factors, although roles have been postulated for both PDGF and TGF-beta in the process of arterial wall wound repair. PDGF is a very potent mitogen for smooth muscle cells, fibroblasts and glial cells. The addition of PDGF to such cells renders the cells competent to enter the cell cycle. A second set of growth factors, termed progression factors, are then believed to be required to progress the cells around the cycle. PDGF addition to cells also elicits a myriad of responses, although it is still not clear if all of these responses are required to elicit the mitogenic response. Structurally, PDGF consists of two non-identical subunits, designated the A and B chains, which are linked by disulfide bonds. Its molecular weight is between about 28-35,000 Daltons, depending on the degree of glycosylation of the sample. Separation of the subunits is believed to result in total loss of biological activity. Recently, it has been suggested that the B chain of PDGF is highly homologous to the predicted protein sequence of the oncogene (v-sis) of simian sarcoma virus (SSV). Indeed, SSV-infected cells either store or secrete a growth factor which is immunologically similar to PDGF. The biosynthesis of this protein has been studied in SSV transformed cells and has been shown to undergo extensive processing, although the major form appears to be a protein of 28,000 Daltons. Two distinct cell surface receptors for PDGF have been identified and are present on smooth muscle cells, fibroblasts, and glial cells. Both PDGF receptors contain tyrosine kinase activity, as has been shown for both the EGF and insulin receptors. It has recently been demonstrated that either the A or the B chain of PDGF is sufficient for mitogenesis. Interestingly, both normal cells and transformed cells have been shown to secrete PDGF-like mitogens into the culture media. It seems likely that growth factors, including PDGF, have some role in normal cellular development, differentiation and tissue repair. The autonomy of transformed cells may be related to endogenous production of growth factors, including PDGF, which may lead to autocrine stimulation and constant stimulation of cell growth.
Fibroblast growth factor (FGF) was initially identified in 1975, Gospodarowicz, D.: J. Biol. Chem., 250:2515-2520 (1975). However, its exact chemical nature has remained obscure until only recently. At least seven (7) forms of FGF have now been identified wherein aFGF and bFGF represent the major forms. One major form is of an acidic nature, i.e., aFGF (pI=5.8), the other major form is basic, i.e., bFGF (pI=9.6). Both forms are present in bovine brain, and the basic form has also been found in bovine pituitary. It is also possible that bFGF may be present in platelets as well. Both species of FGF will stimulate the growth of cells of mesodermal origin, although their potencies are different. The biological effects of FGF reported in the past are now being re-examined, as preparations used in the past were not pure. What is certain, however, is that FGF will stimulate both fibroblast and endothelial growth, as well as repress cell differentiation in cultured muscle cells. No data, however, is believed to be available concerning the biosynthesis of FGF.
The transforming growth factors (both alpha and beta) have very interesting properties. TGF-alpha was first found to be secreted by various transformed cells, and has since been shown to interact with epidermal growth factor (EGF) receptors, and to be structurally (although not antigenically) similar to EGF. TGF-alpha will elicit the same intracellular events as EGF, including cellular proliferation via binding to the EGF receptor. The molecular weights of various species of TFG-alpha vary from about 6,000 to about 11,000 Daltons, and all consist of single polypeptide chains. Recently, a higher molecular weight form (about 25,000 Daltons) of TGF-alpha has been identified in platelets. This may represent a precursor form of other TGF-alphas, although this has not yet been conclusively demonstrated. TGF-alpha, in conjunction with TGF-beta, will allow fibroblasts to grow in soft agar, which is a typical property of transformed cells. Neither TGF-alpha nor TGF-beta individually can do this. TGF-beta has a molecular weight of 25,000 Daltons and consists of a homodimer. The subunits are held together by many disulfide linkages, and destruction of the linkages leads to a loss of biological activity. TGF-beta is also found to be secreted by transformed tissues. Platelets are a major storage site for TGF-beta. Three distinct cell surface receptors for TGF-beta have now been identified by cross-linking studies. The biological effects of TGF-beta are quite complex. The first biological effect noted was the ability of TGF-beta in conjunction with either EGF or TGF-alpha, to stimulate fibroblast growth in soft agar, which is a phenotypic trait of transformed cells. Since then TGF-beta has been shown to also inhibit both normal and transformed cell growth, possibly by lengthening the G.sub.1 phase of the cell cycle, although the target cell density also appears to play an important role in the effect of TGF-beta activity on the cell. TGF-beta will, by itself, stimulate DNA synthesis in serum-deprived, sparse fibroblast cultures. However, TGF-beta will not stimulate DNA synthesis in confluent, density arrested fibroblast cultures. The reason for the distinction has not yet been established. TGF-beta will also affect EGF receptor metabolism. Short-term treatment, i.e., about 1-4 hours, of rat fibroblasts with TGF-beta can decrease the number of high-affinity sites for EGF. Further treatment with TGF-beta results in an overall increase in EGF receptor number for both the low and high-affinity sites. The increase in EGF receptor number by TGF-beta appears to account for a synergistic response to the combination of TGF-beta and EGF, as measured by DNA synthesis in the recipient cells. How these alterations in EGF receptor number are brought about, or the mechanism of synergy between TGF-beta and EGF, are at present unknown.
One problem in studying the effects of PDGF, TGF-alpha and TGF-beta on cells in culture is the difficulty in obtaining large quantities of each factor which is a typical problem associated with proteins generated by cells as indicated above. The major storage site, in normal tissue, for these factors is the platelet. It is not only difficult to obtain large quantities of platelets for large-scale purification, but even if such quantities of platelets could be obtained, platelets presently cannot be used as a practical matter to study the biosynthesis, storage and/or release of these factors. With respect to natural TGF-beta, it can be obtained from bovine kidney (1 kg of kidney will generally yield 3-4 micrograms of TGF-beta) and FGF can be isolated in microgram levels from bovine brain or pituitary. Unfortunately, it is difficult to do biosynthetic studies in these tissues as well. Thus, the establishment of a cell line which can synthesize these factors in generous quantities as well as provide an ample source for the genes and messenger RNAs would be very advantageous for their production and biosynthetic and physiological studies. Certain cell lines (primarily osteosarcomas) have been identified hitherto which are believed to secrete a PDGF-like molecule, Seifert, R. et al: Nature, 311:669-781 (1984); Di Corleto, P. E. et al: Proc. Natl. Acad. Sci. USA, 80:1919-1923, 1983; and Bowen-Pope, D. F. et al: Proc. Natl. Acad. Sci. USA. 81:2396-2400 (1984). However, it is presently believed that to date no cell lines comprised of monophenotypic cloned cells of megakaryocytic lineage and origin, which are believed to be platelet precursors, have been well characterized or established.
The development of an in vitro cell line of megakaryocytic lineage unfortunately has proven to be very difficult. Normal human megakaryocytes can be isolated and grown in tissue culture, Duperray, A. et al: J. Cell Biol., 104:1665 (1987); Mazur, e. et al: Exp. Hematol., 15:340 (1987); Berkow, R. L. et al: J. Lab. Clin. Med., 103:811 (1984); Kimure, H. et al: J. Cell Phys., 118:87-96, 1984; and Tabilo, A. et al: EMBO J., 3:453-459 (1984). But generally, these cultures can only be maintained for short periods of time, and it is difficult to produce large quantities of cells. Some permanent cell lines with megakaryocytic-like features have been suggested, Tabilo, A. et al: EMBO J., 3:453-459 (1984), and Gerwirtz, A. et al: Blood, 60:785-789; however, these cell lines have been derived from patients with nonmegakaryocytic leukemias and show only limited megakaryocytic differentiation. In a recent report by Morgan D. A. et al: J. Cell. Biol., 100:565-573, (1985), they have suggested therein that human cell lines have been developed with properties similar to megakaryocytes. These cell lines, however, are believed to be derived from patients with either various hematologic disorders or normal peripheral blood. They do not show the morphologic features of mature megakaryocytes, though the initial unconfirmed immunohistochemical studies possibly show a population of cells with megakaryoblastic features. These cell lines have been analyzed for cross-reacting material to an antibody directed against PDGF, Pantazis, P. et al: In Cancer Cells, Vol. 3, J. Feramiso, B. Ozanne, and C. Stiles, eds., Cold Spring Harbor laboratory, pp. 153-157, 1985. Intracellular proteins in the range of 12,000-48,000 Daltons were detected, although the mitogenic capability of these proteins has not yet been reported, nor was it reported if this cell line expresses large quantities of these growth factors.
There have also been other recent reports drawn to cell lines alleged to have megakaryocyticlike features. For example, a cell line originating from bone marrow and designated as EST-IU is disclosed in Sledge, G. et al: Cancer Res., 46:2155 (1986); and Roth, B. J. et al: Blood, 72:202 (1988). This EST-IU cell line, however, is not cloned, it is not immortal, i.e., it dies upon continuous culturing, and it has a karyotype of 84. Another cell line designated as DAMI is disclosed in Greenberg, S. M. et al: Blood, 72:1968 (1988). It is reported that the DAMI cell line is originated from circulating blood, it is not a cloned cell line, it expresses the erythroid marker glycophorin, and it has a variable karyotype of 54-64 chromosomes. The cell line designated as CMK has been disclosed in Komatsu, N. et al: Blood, 74:42 (1989). Komatsu, N. et al reports that the CMK is originated from circulating blood, it expresses the monocyte marker OKM5, PMA induces it to express the erythroid marker glycophorin A, and only about 20% of its cells are positive for the gpIIbIIIa protein complex. The literature has also reported the cell line designated as MEG-01 in Ogura, M. et al: Blood, 66:1384 (1985). The MEG-01 cell line is originated from bone marrow, it is not cloned, it expresses the monocyte marker BA-1, and the chromosome number of its cells vary between 56-58. In addition, the MEG-01 cell line has been subsequently cloned into a cell line designated as MEG-01s. However, only about 25% of the cells of the MEG-01s cell line express the gpIIbIIIa protein complex. In addition, the other characteristics of the MEG-01 recited above are featured by the cloned cells of the MEG-01s cloned cell line. A cell line designated as T-33 has also been recently reported by Tange, T. et al: Cancer Res., 48:6137 (1988). The T-33 cell line, however, is originated from blood, it is not cloned, only about 13% of the cells thereof express platelet peroxidase, and it has a karyotype of 51.
Consequently, it would be very desirable to establish xenografts and in vitro cell lines which are monophenotypic for megakaryocytic lineage for providing ample quantities of growth factors, genes and messenger RNAs as well as to assist in the study of megakaryocytes, megakaryocyte associated functions, megakaryopoiesis and megakaryocytic properties expressed by megakaryocytes.